Ebook Heart rate and rhythm molecular basis pharmacological modulation and clinical implications: Part 2

(BQ) Part 2 book Heart rate and rhythm molecular basis pharmacological modulation and clinical implications presents the following contents: Mechanisms of inherited arrhythmia, role of specific channels and transporters in arrhythmia, drugs and cardiac arrhythmia.

Part VMechanisms of Inherited Arrhythmia

.

In cardiology, there are two major clusters of monogenic disorders: (a) thecardiomyopathies due to alterations in sarcomeric and in cytoskeletal proteins,and (b) the arrhythmogenic diseases that are caused by mutations in ion channelsand ion channel-controlling proteins such as the long QT syndromes (LQTS), theBrugada syndromes, the short QT syndromes (SQTS), and the catecholaminergicpolymorphic ventricular tachycardia (CPVT).

N Monteforte and R Bloise

Molecular Cardiology, Fondazione S Maugeri IRCCS, Via Salvatore Maugeri 10/10A, 27100 Pavia, Italy

O.N Tripathi et al (eds.), Heart Rate and Rhythm,

DOI 10.1007/978-3-642-17575-6_21, # Springer-Verlag Berlin Heidelberg 2011 387

Inherited arrhythmogenic diseases are associated with an increased risk forventricular arrhythmias These diseases are often asymptomatic for many yearsand are not detected until the first clinical presentation such as syncope or suddencardiac death In approximately 10–20% of all sudden deaths, no structural cardiacabnormalities can be identified [1] These diseases often affect young, otherwisehealthy individuals, and the conventional electrocardiogram (ECG) is important fordiagnosing established diseases or detecting novel entities associated with suddencardiac death [2 4] The b-blockers are effective in some instances (e.g., LQTS,catecholaminergic ventricular tachycardia) but often an implantable cardioverterdefibrillator (ICD) is the only option for high risk patients.

It is important to consider that the clinical manifestations of these diseases maysignificantly vary from one patient to the other even in the presence of the samegenetic defect In technical terms, this phenomenon is attributed to the “variableexpressivity” (nonuniform clinical severity of carriers of the same genetic defect)and the incomplete penetrance (i.e., the ratio between carriers of a given gene defectand the number of clinically affected individuals is lower than 1) The identification

of the genes underlying the inherited arrhythmogenic syndromes has greatly tributed to the understanding of the substrate for the arrhythmia development, butmore importantly, it has provided major practical information that is helpful whenmanaging affected individuals

con-In this chapter, we focus on the genetic basis, the clinical features, and the maintherapeutic strategies of the most important channelopathies caused by a geneti-cally determined impairment of intracellular calcium handling such as CPVT,Timothy syndrome [(TS), a variant of long QT syndrome (LQT8)], and two geneticvariants of Brugada syndrome (BrS3 and BrS4)

21.2 Catecholaminergic Polymorphic Ventricular

Tachycardia

CPVT is a severe disorder, with a high incidence of sudden cardiac death amongaffected individuals The first report of a patient with this disease was published in

1975 [5], but the first systematic description came in 1978 with the work of Coumel

et al [6] and was further refined by the same group in 1995 [7] In 2001, moleculargenetic studies unveiled that CPVT results from inherited defects of intracellularcalcium handling that cause abnormal Ca2+release form the sarcoplasmic reticulum(SR) We reported for the first time that the autosomal dominant form of the diseasewas caused by mutations in the gene encoding for the cardiac ryanodine receptor(RyR2) [8] Shortly after, the gene for the autosomal recessive form of CPVT wasidentified as the gene encoding cardiac calsequestrin (CASQ2) [9] After identifica-tion of the underlying genetic causes, basic science studies in cell systems andanimal models brought a major advancement to the understanding of arrhythmo-genic mechanisms in this disease

21.2.1 Calcium Handling and Arrhytmogenesis in CPVT

The discovery that genetic defects in Ca2+regulatory proteins such as the ryanodinereceptor (RyR2) [10, 11] and calsequestrin (CASQ2) [12], result in CPVT, hasstimulated many fundamental studies that provided new and compelling evidence

to link abnormal intracellular Ca2+signaling and arrhythmia Calcium that entersthe cell during the plateau phase of the action potential (AP) triggers the release of

Ca2+from SR through ryanodine receptors [13] (Fig.21.1) This process, known as

Ca2+-induced Ca2+release (CICR), amplifies the initial Ca2+entry signal to produce

an elevation of cytosolic Ca2+ [Ca2+]i, triggering the cascade of conformationalchanges leading to contraction of the sarcomere During relaxation, most of the

Ca2+ in the cytosol is recycled into the SR by cardiac SR calcium adenosinetriphosphatase (SERCA2), the activity of which is controlled by phospholamban

CaV1.2

FKBP12.6

JCTN/TRDN RyR2

CASQ2

SR

Plasma Membrane

Fig 21.1 Diagram showing the localization of the proteins involved in the pathogenesis of Ca2+handling SR sarcoplasmic reticulum, NCX sodium–calcium exchanger, JCTN junctin, TRDN triadin SERCA SR calcium adenosine triphosphatase, PLB phospholamban

21 Intracellular Calcium Handling and Inherited Arrhythmogenic Diseases 389

(PLB) Additionally, some of the Ca2+is extruded from the cell by the Na+/Ca2+exchange (NCX) to balance the Ca2+entry.

Spontaneous Ca2+release occurs in the form of self-propagating waves of CICRthat originate locally as spontaneous release events, known as Ca2+ sparks [14].During diastole, individual sparks can lead to local increase in Ca2+current In thepresence of calcium overload, the diastolic Ca2+spark rate and SR channel sensi-tivity to cytosolic Ca2+ increase Spontaneous Ca2+ waves are arrhythmogenicand induce Ca2+-dependent depolarizing currents, thereby causing oscillations

of the membrane potential known as delayed afterdepolarizations (DAD) [15].When sufficiently large, DADs evoke extrasystolic APs, thereby causing triggeredarrhythmias

Substantial evidence supports the concept that changes in luminal Ca2+ute to termination of CICR and facilitate RyR2 to enter in a refractory state thatsuppresses diastolic Ca2+ release Alterations in luminal Ca2+ control of Ca2+release are, therefore, expected to lead to serious disruptions of the cellular Ca2+cycling

contrib-Alternative hypotheses have been advanced to explain the functional quences of RyR2 mutations CPVT-associated mutations may lead to abnormaldissociation (reduced binding affinity) of the auxiliary protein FKBP12.6 fromRyR2 [16] Less RyR2-FKBP12.6 binding in turn influences channel gating causingincreased diastolic Ca2+leak from the sarcoplasmic reticulum (SR), a phenomenonknown to favor the onset of DADs and arrhythmias Alternatively, mutations maychange RyR2 sensitivity to luminal Ca2+, thus reducing the Ca2+threshold requiredfor generation of spontaneous Ca2+release [17].CASQ2 mutations in the autosomalrecessive form of CPVT also result in deregulated SR Ca2+release and arrhythmo-genic DADs [18–22] This effect is due to reduced Ca2+ buffering properties ofCASQ2 and/or by loss of CASQ2-mediated RyR2 regulation Irrespective of which

conse-of these mechanisms is involved, the final effect is the generation conse-of genic spontaneous Ca2+release from the SR and generation of DADs

arrhythmo-21.2.2 Genetic Bases of CPVT

Most familial CPVTs show autosomal dominant pattern of inheritance In 1999,Swan et al [23] identified a significant linkage between the CPVT phenotype andmicrosatellite markers at locus 1q42-q43 Based on this information, we performedmolecular screening and identified cardiac RyR2 as the mutant CPVT gene [8].Involvement of RyR2 in the genesis of CPVT was subsequently confirmed byseveral other investigators (http://www.fsm.it/cardmoc) A recent analysis of pub-lished RyR2 mutations shows that they tend to cluster in 25 exons encoding 4discrete domains of RyR2 protein: domain I (amino acid (AA) 77–466), II (AA2246–2534), III (AA 3778–4201), and IV (AA 4497–4959) (DI-DIV) Theseclusters are composed of amino acid sequences highly conserved through speciesand among RyR isoforms [24] and are thought to be functionally important

Soon after identification of theRyR2 mutations in the autosomal dominant form

of CPVT, Lahat et al [9] mapped the recessive variant on chromosome 1p23-21and subsequently identified one mutation on theCASQ2 gene, encoding for cardiaccalsequestrin.CASQ2 mutations represent only 1–2% of all genotyped CPVTs.More recently, based on the evidence that the patients with Andersen–Tawilsyndrome may develop bidirectional ventricular tachycardia [25, 26], i.e., thetypical arrhythmia observed in CPVT, it has been suggested that some CPVTcases can be explained byKCNJ2 mutations (phenocopy) In 2007, a new autosomalrecessive form of CPVT mapping on the chromosomal locus 7p14-22 was reported

by Bhuiyan et al [27], but the responsible gene has not yet been discovered

So far, more than 70 different mutations have been associated with CPVT, andthese are all single-base pair substitutions causing the substitution of an amino acid

As expected for autosomal recessive disorders, the number of families with CPVTlinked toCASQ2 mutations is fairly small At present, only seven mutations havebeen discovered, and they can be inherited in homozygous or compound heterozy-gous form A recent analysis from our group [28] has demonstrated that geneticscreening on theRyR2 gene is able to identify at least 60–65% of patients with theclinical phenotype; therefore, genetic screening should be recommended since it isable to identify most of the affected subjects and could then be extended to familymembers

21.2.3 Mechanisms of Arrhythmias in Autosomal Dominant

CPVT

The RyR2 is a tetrameric channel that regulates the release of Ca2+from SR tothe cytosol during the plateau phase of the cardiac AP When RyR2 activity ismodified/altered leading to an increase or reduction of the amount of Ca2+released,both the SR and the cytosolic Ca2+ concentration may be affected This inducescompensatory phenomena that tend to restore the cellular calcium balance, such asthe activation of the cardiac NCX Unfortunately, such compensatory mechanismsmay be arrhythmogenic RyR2 function (SR Ca2+release) is regulated by severalaccessory proteins, such as CASQ2, triadin, junctin, and FKBP12.6 (Fig.21.1).Furthermore, the adrenergic tone controls the RyR2 channel through phosphoryla-tion, which is a crucial step determining the amount of Ca2+ released from SR.Catecholamines activate protein kinase-A (PKA) and calcium-calmodulin depen-dent kinase II (CaMKII) that phosphorylates RyR2 at different sites and acts as athrottle on the Ca2+release process [29]

21.2.3.1 RyR2 Mutations and CPVT

The effects of RyR2 mutations have been studied in vitro and in vivo usingdifferent experimental models RyR2 mutations can affect both the activation

21 Intracellular Calcium Handling and Inherited Arrhythmogenic Diseases 391

and the inactivation of the channel in several ways It is noteworthy that, whenviewed independently from the subcellular mechanisms, the final common effect ofCPVT mutations (both RyR2 and CASQ2) appears similar to that of digitalisintoxication, viz Ca2+overload, activation of NCX in the forward mode, generation

of transient inward NCX current (Iti), and delayed after-depolarizations (DADs).The proposed “primum movens” leading to Ca2+overload is the uncontrolled

Ca2+release (leakage) during diastole, which is mainly detectable upon adrenergicactivation [30] (phosphorylation); but according to different authors, it may already

be present in the unstimulated conditions [31,32] Given the complexity of the SR

Ca2+release process, the leakage could in principle be due to several mechanisms[16,30,31]

21.2.3.2 RyR2–CPVT Mouse Models

Knock-in mouse models have been pivotal to the understanding of the cellular andwhole-heart pathophysiology of CPVT [33–35] Based on the assumption that byengineering RyR2–CPVT mutation in the mouse genome, it is possible to repro-duce the phenotype observed in the clinical setting, the initial evidence wasprovided by our group in 2005 By homologous recombination, we created aconditional knock-in mouse harboring the R4496C mutation This is the firstmutation that we identified in CPVT patients and it is present in several unrelatedCPVT families [34] R4496C mice develop typical CPVT bidirectional VT in theabsence of structural abnormalities [34] This model has been instrumental todemonstrated adrenergic-dependent DADs, increased NCX-transient inward cur-rent (Iti), and triggered activity as the cellular mechanisms for CPVT [36](Fig.21.2) In a subsequent study [37], we observed the onset of abnormal Ca2+waves during diastole, which paralleled the occurrence of DAD development both

at baseline and during isoproterenol superfusion Increased propensity to DADdevelopment in RyR2-R4496C mice was also demonstrated in isolated Purkinje

Fig 21.2 DADs recorded

from an isolated RyR2R4496C+/

myocyte stimulated at 1–5 Hz.

Note that DAD amplitude

increases and DAD coupling

interval decreases at faster

pacing frequencies (modified

from [ 36 ])

cells by Cerrone et al [38] Finally, additional data supporting the concept thatDAD-mediated triggered activity is the arrhythmogenic mechanism for CPVT wereprovided by Paavola et al [39], who recorded DADs using monophasic APs inCPVT patients.

In an optical mapping study in collaboration with Dr Jalife and his coworkers,

we showed that both polymorphic and bidirectional VT have a focal origin [38].Epicardial optical mapping was used to demonstrate that during bidirectional VT,the ventricular beats alternatively originate from the right and from the left ventricleand arise from an area coincident with the anatomic insertion of the major bundlebranches of the conduction system Interestingly, administration of Lugol’s solutionthat ablates the Purkinje network is able to convert bidirectional VT to monomor-phic left-sided VT In the same study, endocardial optical maps also showed thatduring polymorphic VT the site of origin of the beats mapped on the endocardialright ventricular wall correspond to free running Purkinje fibers Overall theseexperiments support the relevant role of Purkinje network in the pathogenesis ofarrhythmias in CPVT

21.2.4 Mechanisms of Arrhythmias in Autosomal Recessive

CPVT

CASQ2 has been initially described as a Ca2+buffering protein resident in the SRlumen and exists in monomeric and polymeric forms When luminal [Ca2+] is low,CASQ2 binds to junctin and triadin and inhibits SR calcium release from RyR2.Conversely, in the presence of a rise in luminal SR[Ca2+], the binding betweenCASQ2, triadin, and junctin is weakened and the open probability for RyR2increases [40] Overall evidence concurs to attribute to CASQ2 the roles of a

Ca2+buffer molecule and a RyR2 modulator

21.2.4.1 CASQ2 Mutations and CPVT

Mutations inCASQ2 that cause the autosomal recessive CPVT are rather mon, and so far no phenotypical differences have been identified between CASQ2-and RyR2–CPVT The fewCASQ2 mutations reported so far have been extensivelystudied in in-vitro and in transgenic animal MODELS In vitro studies have high-lighted that the mutations may lead to major alterations in CASQ2 functions as theymay impair CASQ2 polymerization, alter its buffering properties, and modifyCASQ2–RyR2 interaction Terentyev et al [17] suggested that a reduction orabsence of CASQ2, as it happens with the truncation mutants, leads to a decrease

uncom-of the time necessary to reestablish Ca2+ storage, thus facilitating a prematureactivation of RyR2 and, as a consequence, diastolic Ca2+leakage

21 Intracellular Calcium Handling and Inherited Arrhythmogenic Diseases 393

21.2.4.2 CASQ2–CPVT Mouse Models

As in the case of RyR2–CPVT, mouse models reproducing the autosomal recessiveCASQ2–CPVT have provided important pathophysiological information buthave also been of great value for the unraveling of some molecular mechanisms

of cardiac Ca2+ regulation Knollmann et al [21] created a CASQ2 knock-outmouse model, in which VT and ventricular fibrillation (VF) could be induced byb-adrenergic stimulation (isoproterenol) or even acute stressors such as auditorystimuli In isolated CASQ2 null myocytes, the authors observed increased diastolic

Ca2+leakage leading to DADs and triggered activity, thus proving that DADs arethe common final arrhythmogenic mechanisms in RyR2- and CASQ2–CVPT.More recently, we developed theCASQ2–R33Q/R33Q knock-in mouse model thatreproduces the typical CPVT phenotype [41] At variance with theRyR2–R4496Cmodel, arrhythmias in these mice occur in the presence of mild stressors (Fig.21.3).CASQ2–R33Q/R33Q cardiomyocytes showed DADs and triggered activity notonly during b-adrenergic stimulation but also in resting conditions [41] Interest-ingly, we observed a prominent reduction of CASQ2 in our mice with the R33Qmutation and we were able to show that mutant calsquestrin is prone to increasedtrypsin degradation On the basis of these observations, it is possible to speculatethat the key mechanism for autosomal recessive CPVT is the reduction in the

cellular content of caslequestrin that leads to an increased propensity for diastolic

Ca2+leak

21.2.5 Clinical Presentation and Diagnosis

Patients with CPVT typically present with stress-induced syncope and/or suddencardiac death [7, 8] Symptoms can occur in early childhood [42] and the meanage of onset of the first syncope in our large cohort of CPVT [43], and recentlyconfirmed in an additional series [44], is 12 years In the absence of treatment, thedisease is highly lethal, with an estimated incidence of sudden death of 30% beforeage 40 [45] Growing evidence shows that sudden death may be the first manifesta-tion of the disease, making prevention of lethal events a difficult task

Individuals with CPVT show an unremarkable ECG, which also makes thediagnosis difficult Frequently, CPVT patients seek medical attention for the evalu-ation of unexplained syncope; in this setting, very often they are misdiagnosed asbeing affected by vasovagal syncope or epilepsy since resting ECG is normal.Minor, nondiagnostic features at rest are sinus bradycardia and prominent Uwaves [46] (Fig 21.4) Some authors also reported sinus bradycardia in some

Fig 21.4 Resting ECG in a

CPVT patient showing a low

heart rate and prominent U

wave

21 Intracellular Calcium Handling and Inherited Arrhythmogenic Diseases 395

CPVT patients [7,47] and Postma et al [47] hypothesized that bradycardia mayresult from impaired Ca2+handling by mutant RyR2 channels in sinoatrial nodalcells The presence of prominent U waves has also been reported, but its diagnosticvalue has never been systematically evaluated or demonstrated Furthermore, amild QT prolongation in some CPVT cases was reported [6, 7]; thus CPVTdifferential diagnosis should include LQTS LQTS patients with a mild phenotype(borderline QT interval and no symptoms) do exist, but their prognosis iscompletely different from that of CPVT, which presents a higher incidence ofsudden death and a limited response to b-blocker therapy [42,48].

Independently from the clinical presentation (syncope or aborted sudden death),the most important clinical test to diagnose CPVT is the exercise stress test Indeed,

in clinically overt CPVT (penetrant cases), there is a highly reproducible pattern ofarrhythmias evoked during exercise stress test or isoproterenol infusion [42,49].These observations enforce the concept that an exercise stress test should beperformed in the routine evaluation of unexplained syncope, especially if adrener-gic trigger is evident The typical behavior of CPVT arrhythmias is that of aprogressive worsening upon increase in workload: isolated premature beats orcouplets initially appear between 90 and 110 bpm followed by runs of nonsustained

or sustained VT when heart rate further increases [50] (Fig.21.5) Supraventriculararrhythmias are also a common finding and often precede the onset of ventricular

arrhythmias [51] The morphology of VT is often the hallmark of the disease: theso-called bidirectional VT [8, 42] which is characterized by a 180 beat-to-beat

rotation of the axis of the QRS complexes on the frontal plane (Fig.21.6) Althoughthis pattern is recognizable in the majority of patients, it is important to be awarethat some patients also present irregular polymorphic VT Furthermore, the initialpresentation of the disease may also be that of a VF triggered by sudden adrenergicactivation [42] that may be interpreted as idiopathic VF in the absence of docu-mentation of typical CPVT arrhythmias Holter monitoring and implantable looprecorders may be helpful for diagnosis in such instances and especially for thosepatients in whom emotional triggers (alone or in combination with exercise) is morearrhythmogenic than exercise alone [52,53]

Programmed electrical stimulation (PES) does not contribute to the clinicalevaluation of CPVT patients since ventricular arrhythmias are rarely inducible inCPVT; conversely epinephrine infusion may often induce the typical pattern of VT,although its diagnostic sensitivity does not appear to be higher than that of exercisestress test

In 2002, we reported data showing that exercise/emotion-induced syncopeoccurs in 67% of patients, while in 33% of families juvenile SCD was detectable[42] These data were substantially confirmed by a Japanese group in 2003 [51], byanother European study in 2005 [47], and by a follow-up reanalysis of our database

on 119 patients (which showed that close to 80% of patients experience cardiacevents before 40 years of age) [45] Overall, approximately 30% of the patientshave a first syncope or cardiac arrest before 10 years of age, and death or abortedcardiac arrest occurring with an incidence close to 20% up to 20 years of age

An additional hallmark of severity is the low percentage (20%) of asymptomaticcarriers of mutations in theCPVT genes (high penetrance) [42,47] Therefore, onthe basis of current data, CPVT should be regarded as one of the most severe amongthe inherited arrhythmogenic disorders

21.2.6 Current Therapy and Future Directions

21.2.6.1 CPVT Therapy in the Clinical Setting

Based on the evidence of the critical role of adrenergic stimulation as a trigger forarrhythmias, b-blockers were proposed as the mainstay of CPVT therapy since the

Fig 21.6 Typical bidirectional ventricular tachycardia in a CPVT patient

21 Intracellular Calcium Handling and Inherited Arrhythmogenic Diseases 397

earlier reports [6,7] and are indeed indicated both for chronic treatment as well asacute therapy of sustained ventricular tachycardia b-blockers should be startedimmediately when CPVT is diagnosed; agreement in the scientific communityseems to indicate nadolol as the first choice among the available options – for itsonce daily dosing and nonselective inhibition of adrenergic stimuli Asymptomaticbradycardia in these patients should not be considered as a reason to reduce thedosage of b-blocker therapy In fact, the demonstration that DADs induce triggeredactivity and that DAD-induced arrhythmias are facilitated by faster heart rates,provides a rationale to consider the bradycardic effect of b-blockers, an additionalantiarrhythmic benefit, along with the inhibition of sympathetic drive [54].There are conflicting evidences on the long-term effectiveness of b-blockers inthe published reports Although Leenhardt [7] and Postma [47] have reported analmost complete prevention from the recurrence of cardiac events with the excep-tion of noncompliant patients, we [42] and others [44,49] observed recurrences ofcardiac events or incomplete protection from exercise-induced arrhythmias inCPVT patients treated with the maximally tolerated dose In the Italian CPVTRegistry, the incidence of recurrent arrhythmia while on therapy is as high as 30%[42] In case of recurrences of syncopal episodes or VT while on therapy, theimplant of an ICD should be considered Obviously, after ICD implant b-blockertreatment should be maintained to minimize the risk of device interventions.Furthermore, in our series, 50% of implanted patients received an appropriatedevice intervention in a 2-year follow-up [45] The incomplete protection afforded

by b-blockers calls for the need to identify adjunctive affective therapies

Calcium channel blockers (CCB), in particular verapamil, have been studied bydifferent groups in a limited patient series as a possible alternative to b-blockertherapy by Swan et al [55] and Sumitomo et al [49] The study of Rosso et al [56]evaluated the efficacy of a combined association between b-blockers and verapa-mil: in their series, the combination of therapies reduced or even suppressed therecurrences of exercise-induced arrhythmias and/or ICD shocks More recently,Watanabe et al [57] reported a previously unrecognized inhibitory action offlecainide on RyR2 channels, which, together with flecainide’s inhibition of Na+channels, was able to prevent CPVT in two individuals that had remained highlysymptomatic on conventional drug therapy Wilde et al [58] provided preliminaryevidence for a long-term effectiveness of left cardiac sympathetic denervation(LCSD) in three CPVT patients, and Scott et al [59] reported a case of successfulbilateral thorascopic sympathectomy, but patients with recurrences of sustained VTand syncope on b-blockers and LCSD are present in our cohort of CPVT patients

21.2.6.2 Experimental Therapies for CPVT

Experiments in cell systems and CPVT animal models have been carried out toexplore new therapeutic possibilities The attempt to use FKBP12.6 stabilizingdrugs [S107 [33] or K201 [60]] yielded conflicting results Another interestingapproach is that of inhibiting the effects of b-adrenergic stimulation by acting on

the downstream targets of RyR2 phosphorylation The pharmacological inhibition

of CAMKII (that phosphorylates RyR2 during adrenergic activation) is a promisingapproach CAMKII phosphorylates RyR2 at different sites Moreover, it is knownthat CAMKII inhibition reduces diastolic Ca2+ leakage and NCX-Iti [61] thatgenerates DADs Preliminary observations from our group in theRyR2-R4496C/

WT mouse model suggest that a specific CAMKII inhibitor, KN93 [62], couldprevent arrhythmias both in vitro and in vivo, thus providing encouraging datatoward novel therapeutic strategies involving this pathway

Timothy syndrome (TS) is a recently described variant of long QT syndrome(LQT8) that results from mutations present in the gene encoding for the L-typecalcium channel (Cav1.2) It is considered a very rare and malignant form of LQTS,with very high lethality The high lethality in some cases is not related to the cardiacphenotype, but to extracardiac problems Indeed, in addition to excessive prolonga-tion of QT interval, patients affected by TS have multi-organ disorders includinglethal arrhythmias, congenital heart diseases, syndactyly, development delay, met-abolic disturbance, immunodeficiency, and autism

21.3.1 L-Type Calcium Channel

The pore forming a1 protein responsible for L-type Ca2+channel (LTCC) in heart isidentified as Cav1.2 [63] This channel is made up of a1, a2/d, and b subunits Thea1 subunit forms the ion-selective pore, the voltage sensor, the gating machinery,and the binding sites for channel-modulating drugs b, a2, and d subunits appear tohave a regulatory effect [64, 65] (Fig 21.7) Cav1.2 is the major Ca2+ channel

S1

coo Intracellular Extracellular

-NH3 +

S6

Fig 21.7 Diagram showing predicting topology of L-type Ca2+channel

21 Intracellular Calcium Handling and Inherited Arrhythmogenic Diseases 399

expressed in ventricular myocytes It produces a voltage-dependent inward Ca2+current (ICaL) that activates upon depolarization and it is a crucial player in themaintenance of the plateau of cardiac AP Ca2+ ions play an important role inexcitation–contraction coupling, andICais the critical trigger for the release of Ca2+ions from the sarcoplasmic reticulum to initiate contraction Therefore, any pertur-bation of LTCC is likely to induce arrhythmias Protein kinase A (PKA), proteinkinase C (PKC), and Ca2+-binding protein calmodulin constitute key mechanismsthat control Ca2+influx Furthermore, Cav1.2 channel activity is also enhanced by

Ca2+, catecholamines, and CaMKII [66]

21.3.2 Genetic Basis of Timothy Syndrome

In 2004, we identified the G1216A transition in exon 8A (an alternatively splicedexon) inCACNA1C, which caused the G406R amino acid replacement in DI/S6 in

TS patients [67] In 2005, Splawski et al reported two individuals with a severevariant of TS but without syndactyly; they named this form of the disease asTimothy Syndrome type 2 (TS2) [68] Genetic analyses showed two mutationsG1216A and G1204A in exon 8, which caused G406R and G402S amino acidtransitions, respectively Exons 8 and 8A are mutually exclusive as they encode thesame structural domain (DI/S6), but one of the two must be present to encode afunctional channel Familial recurrence of TS phenotype is rare Functional in vitrocharacterization of G406R mutation suggested that the mutation leads to anincrease of inwardICadue to loss of voltage-dependent inactivation APs are likely

to be significantly prolonged as a consequence of this TS mutation and DADs and,therefore, triggered activities are likely to be the electrophysiological mechanismsfor arrhythmias in this disease

21.3.3 Clinical Presentation and Diagnosis

The most apparent features of this syndrome are the extreme prolongation of QTinterval (Fig.21.8) associated with lethal arrhythmias and syndactyly, which mayprovide clues for preliminary diagnosis [69,70] However, other cardiac or noncar-diac manifestations, including congenital heart disease, facial dysmorphisms, neu-ropsychiatric disorders, metabolic disturbances, immunodeficiency, and recurrentinfection are also common, but may not simultaneously occur in the same TSpatient Arrhythmic events (TdP, VT, VF, or SCD) represent the most relevantcause of death in TS patients, but several other features contribute to the TSphenotype: congenital heart disease (PDA, PFO, ToF); hypertrophic cardiomyopa-thy and ventricular systolic dysfunction; hand/feet syndactyly; facial dysmorph-isms; predisposition to sepsis; metabolic (severe hypoglycemia) and immunologic

(recurrent infections) disturbances; neuropsychiatric involvement (autism, seizures,psychological developmental delays).

CACNA1C is highly expressed in adult heart and its mRNA is also widelyexpressed in multiple adult and fetal tissues, including brain, gastrointestinalsystem, lung, immune system, smooth muscle and testis This may explain why a

TS patient has both cardiac and extracardiac disorders even at birth At present,most of the TS patients have been treated with b-blockers as it is considered agenerally effective therapy in patients with congenital LQTS Additional pharma-cological therapies (mexiletine, CCBs) have been proposed in an attempt to shortenventricular repolarization, restore 1:1 conduction, and reduce the risk of arrhyth-mias but their use has to be considered to be still in an experimental evaluationphase The implantable defibrillator is an alternative for patients who remain at riskfor cardiac arrest despite pharmacological therapy

Finally, it is important to note that due to extensive multiorgan involvement in

TS, the patients may also die due to other causes such as severe infections probablyrelated to an altered immune response and intractable hypoglycemia

Brugada syndrome (BrS) is an inherited cardiac arrhythmogenic disorder thatwas described as a clinical entity in 1992 [71] It is considered a “primary electricaldisease,” occurring mostly in the absence of overt structural abnormalities Theelectrocardiographic diagnostic feature of the disease is the presence of an ST

Fig 21.8 Resting ECG in a patient with Timothy syndrome showing an important QT prolongation

21 Intracellular Calcium Handling and Inherited Arrhythmogenic Diseases 401

segment elevation 2 mm in, at least, two of the three right precordial leads(V1–V3) [72], with a “coved morphology” and with incomplete or completeright bundle branch block (Fig 21.9) The syndrome is associated with anincreased risk of SCD among affected patients The age of onset of clinicalmanifestations is the third to fourth decade of life, and male gender is associatedwith a more malignant form of the disease.

At the present time, no pharmacological therapy has proven effective in ing survival in BrS patients Therefore, clinicians should risk stratify patients todecide whether an implantable defibrillator is needed At present, the accuracy ofrisk stratification is rather poor Conflicting evidence exists on the prognostic value

improv-of PES and no other predictors improv-of adverse outcome are available A consensus exists

on the indication of an ICD in cardiac arrest survivors Patients with a spontaneouspattern and history of syncope are at higher risk of cardiac arrest, and they should beregarded as candidates for an ICD Patients with a spontaneous ST segment eleva-tion without history of syncope present an intermediate risk of sudden cardiacdeath Finally, patients with a negative phenotype or who have a diagnostic ECGonly after receiving a pharmacological challenge, consisting of intravenous admin-istration of Na+channel blocking agents, are at lower risk of cardiac events [73] Insymptomatic patients, the treatment of choice is the ICD

The initial identification of mutations in cardiac Na+ channel, SCN5A, waspublished in 1998 [74] and several SCN5A mutations in BrS have now beenreported (http://www.fsm.it/cardmoc) However,SCN5A mutations account for nomore than 20% of clinically diagnosed BrS cases Another gene,GPD1-L, encodingfor the glycerol-3-phosphate-dehydrogenase 1-like protein, has also been linkedwith the BrS Recently, mutations in genes encoding the cardiac LTCC a1 subunit

Fig 21.9 Resting ECG in

a patient with Brugada

syndrome showing “coved”

ST segment elevation

(CACNA1C) and the b2b subunit (CACNB2) have been associated with a clinicalentity encompassing a BrS phenotype combined with short QT intervals [2].Antzelevitch et al [2] demonstrated an association between loss-of-function muta-tions in the a1and b2bsubunits of LTCC and the BrS phenotype (defined as BrStypes 3 and 4, respectively).

Defective Na+ channels amplify the heterogeneity in electrical characteristicsamong different transmural cell types and result in voltage gradients betweenepicardium and endocardium that drive an electrotonic current causing ST segmentelevations and arrhythmias based on transmural phase 2 reentry By analogy, it hasbeen suggested that a loss-of-function in LTCC activity, secondary to mutations inCACNA1C and CACNB2, may create arrhythmogenic transmural dispersion due tothe preferential abbreviation of right ventricular epicardial APs Little is knownabout outcome of patients affected by this new type of BrS, and further studies areneeded to characterize the clinical features of these patients

21.5 Conclusions

Although mutations only in the genes encoding for Na+and K+channels had beenimplicated in the genesis of inherited arrhythmogenic syndromes for several years,there is now evidence that proteins controlling intracellular Ca2+abnormalities play

a major role in determining genetically determined arrhythmogenic substrates Ofparticular impact in the field has been the discovery of mutations inRyR2 Thisdiscovery has opened the field of what we could call the “sarcoplasmic reticulumdiseases.” Besides RyR2, CASQ2 has also been implicated in arrhythmogenesis,and it is likely that other proteins of the RyR2 macromolecular complex and/oradditional sarcoplasmic proteins that concur to regulate intracellular Ca2+will beadded to the list of proteins that cause inherited arrhythmias

Finally, we now know that mutations in genes encoding different subunits of theL-type Ca2+channel may cause different arrhythmogenic diseases The paradigm ofopposite phenotypes associated with loss-of-function vs gain-of-function muta-tions, identified in cardiac channelopathies, holds true for mutations affectingLTCCs Interestingly, both gain-of-function mutations that cause Timothy syn-drome and loss-of-function mutations that cause an overlapping syndrome combin-ing Brugada syndrome and Short QT syndrome seem to be very rare, suggestingthat the vital role of the Ca2+channel may tolerate few mutations and be otherwisenoncompatible with life

One fascinating aspect of mutations that alter cardiac cellular Ca2+homeostasis

is that they open the scope for very interesting investigations about possiblemethods to counteract these dysfunctions by acting on several molecular targets.The effort to devise novel therapeutic strategies for severe phenotypes associatedwith intracellular calcium handling abnormalities is the next challenge for clini-cians and basic scientists in this field

21 Intracellular Calcium Handling and Inherited Arrhythmogenic Diseases 403

Loss-3 Gussak I, Brugada P, Brugada J, Wright RS, Kopecky SL, Chaitman BR, et al Idiopathic short

QT interval: a new clinical syndrome? Cardiology 2000;94:99–102.

4 Haissaguerre M, Sacher F, Derval N, Jesel L, Deisenhofer I, de Roy L, et al Early tion in the inferolateral leads: a new syndrome associated with sudden cardiac death J Interv Card Electrophysiol 2007;18:281.

repolariza-5 Reid DS, Tynan M, Braidwood L, Fitzgerald GR Bidirectional tachycardia in a child A study using His bundle electrography Br Heart J 1975;37:339–44.

6 Coumel P, Fidelle J, Lucet V, Attuel P, Bouvrain Y Catecholamine-induced severe cular arrhythmias with Adam-Stokes in children: report of four cases Br Heart J 1978; 40(Suppl):28–37.

ventri-7 Leenhardt A, Lucet V, Denjoy I, Grau F, Ngoc DD, Coumel P Catecholaminergic phic ventricular tachycardia in children A 7-year follow-up of 21 patients Circulation 1995; 91:1512–9.

polymor-8 Priori SG, Napolitano C, Tiso N, Memmi M, Vignati G, Bloise R, et al Mutations in the cardiac ryanodine receptor gene (hRyR2) underlie catecholaminergic polymorphic ventricular tachycardia Circulation 2001;103:196–200.

9 Lahat H, Pras E, Olender T, Avidan N, Ben-Asher E, Man O, et al A missense mutation in a highly conserved region of CASQ2 is associated with autosomal recessive catecholamine- induced polymorphic ventricular tachycardia in Bedouin families from Israel Am J Human Genet 2001;69:1378–84.

10 Napolitano C, Priori SG Diagnosis and treatment of catecholaminergic polymorphic cular tachycardia Heart Rhythm 2007;4:675–8.

ventri-11 Laitinen PJ, Swan H, Piippo K, Viitasalo M, Toivonen L, Kontula K Genes, exercise and sudden death: molecular basis of familial catecholaminergic polymorphic ventricular tachy- cardia Ann Med 2004;36 Suppl 1:81–6.

12 Eldar M, Pras E, Lahat H A missense mutation in the CASQ2 gene is associated with autosomal-recessive catecholamine-induced polymorphic ventricular tachycardia Trends Cardiovasc Med 2003;13:148–51.

13 Bers DM Cardiac excitation–contraction coupling Nature 2002;415:198–205.

14 Venetucci LA, Trafford AW, O’Neill SC, Eisner DA The sarcoplasmic reticulum and arrhythmogenic calcium release Cardiovasc Res 2008;77:285–92.

15 Pogwizd SM, Bers DM Cellular basis of triggered arrhythmias in heart failure Trends Cardiovasc Med 2004;14:61–6.

16 Wehrens XH, Lehnart SE, Huang F, Vest JA, Reiken SR, Mohler PJ, et al FKBP12.6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death Cell 2003;113:829–40.

17 Jiang D, Wang R, Xiao B, Kong H, Hunt DJ, Choi P, et al Enhanced store overload-induced Ca2+ release and channel sensitivity to luminal Ca2+ activation are common defects of RyR2 mutations linked to ventricular tachycardia and sudden death Circ Res 2005;97: 1173–81.

18 Terentyev D, Viatchenko-Karpinski S, Gyorke I, Volpe P, Williams SC, Gyorke S questrin determines the functional size and stability of cardiac intracellular calcium stores: mechanism for hereditary arrhythmia Proc Natl Acad Sci USA 2003;100:11759–64.

19 Terentyev D, Nori A, Santoro M, Viatchenko-Karpinski S, Kubalova Z, Gyorke I, et al Abnormal interactions of calsequestrin with the ryanodine receptor calcium release channel complex linked to exercise induced sudden cardiac death Circ Res 2006;98:1151–8.

20 di Barletta MR, Viatchenko-Karpinski S, Nori A, Memmi M, Terentyev D, Turcato F, et al Clinical phenotype and functional characterization of CASQ2 mutations associated with catecholaminergic polymorphic ventricular tachycardia Circulation 2006;114:1012–9.

21 Knollmann BC, Chopra N, Hlaing T, Akin B, Yang T, Ettensohn K, et al CASQ2 deletion causes sarcoplasmic reticulum volume increase, premature Ca2+-release, and catecholaminergic polymorphic ventricular tachycardia J Clin Invest 2006;116:2510–20.

22 Dirksen WP, Lacombe VA, Chi M, Kalyanasundaram A, Viatchenko-Karpinski S, Terentyev

D, et al A mutation in calsequestrin, CASQ2 D307H, impairs sarcoplasmic reticulum Ca 2+ handling and causes complex ventricular arrhythmias in mice Cardiovasc Res 2007;75: 69–78.

-23 Swan H, Piippo K, Viitasalo M, Heikkil €a P, Paavonen T, Kainulainen K, et al Arrhythmic disorder mapped to chromosome 1q42-q43 causes malignant polymorphic ventricular tachy- cardia in structurally normal hearts J Am Coll Cardiol 1999;34:2035–42.

24 George CH, Jundi H, Thomas NL, Fry DL, Lai FA Ryanodine receptors and ventricular arrhythmias: emerging trends in mutations, mechanisms and therapies J Mol Cell Cardiol 2007;42(1):34–50.

25 Plaster NM, Tawil R, Tristani-Firouzi M, Canu´n S, Bendahhou S, Tsunoda A, et al Mutations

in Kir2.1 cause the developmental and episodic electrical phenotypes of Andersen’s syndrome Cell 2001;105:511–9.

26 Tristani-Firouzi M, Jensen JL, Donaldson MR, Sansone V, Meola G, Hahn A, et al Functional and clinical characterization of KCNJ2 mutations associated with LQT7 (Andersen syndrome).

J Clin Invest 2002;110:381–8.

27 Bhuiyan ZA, Hamdan MA, Shamsi ET, Postma AV, Mannens MM, Wilde AA, et al A novel early onset lethal form of catecholaminergic polymorphic ventricular tachycardia maps to chromosome 7p14-p22 J Cardiovasc Electrophysiol 2007;18:1060–6.

28 Bai R, Napolitano C, Bloise R, Monteforte N, Priori S Yield of genetic screening in inherited cardiac channelopathies: how to prioritize access to genetic testing Circ Arrhythmia Electro- physiol 2009;2:6–15.

29 Stern MD, Cheng H Putting out the fire: what terminates calcium-induced calcium release in cardiac muscle? Cell Calcium 2004;35:591–601.

30 George CH, Higgs GV, Lai FA Ryanodine receptor mutations associated with stress-induced ventricular tachycardia mediate increased calcium release in stimulated cardiomyocytes Circ Res 2003;93:531–40.

31 Jiang D, Xiao B, Zhang L, Chen SR Enhanced basal activity of a cardiac Ca2+release channel (ryanodine receptor) mutant associated with ventricular tachycardia and sudden death Circ Res 2002;91:218–25.

32 Jiang D, Xiao B, Yang D, Wang R, Choi P, Zhang L, et al RyR2 mutations linked to ventricular tachycardia and sudden death reduce the threshold for store-overload-induced

Ca2+release (SOICR) Proc Natl Acad Sci USA 2004;101:13062–7.

33 Lehnart SE, Mongillo M, Bellinger A, Lindegger N, Chen BX, Hsueh W, et al Leaky Ca2+release channel/ryanodine receptor 2 causes seizures and sudden cardiac death in mice J Clin Invest 2008;118:2230–45.

34 Cerrone M, Colombi B, Santoro M, di Barletta MR, Scelsi M, Villani L, et al Bidirectional ventricular tachycardia and fibrillation elicited in a knock-in mouse model carrier of a mutation in the cardiac ryanodine receptor Circ Res 2005;96:e77–82.

35 Kannankeril PJ, Mitchell BM, Goonasekera SA, Chelu MG, Zhang W, Sood S, et al Mice with the R176Q cardiac ryanodine receptor mutation exhibit catecholamine-induced ventricular tachycardia and cardiomyopathy Proc Natil Acad Sci USA 2006;103: 12179–84.

21 Intracellular Calcium Handling and Inherited Arrhythmogenic Diseases 405

36 Liu N, Colombi B, Memmi M, Zissimopoulos S, Rizzi N, Negri S, et al Arrhythmogenesis in catecholaminergic polymorphic ventricular tachycardia: insights from a RyR2 R4496C knock-

in mouse model Circ Res 2006;99:292–8.

37 Fernandez-Velasco M, Rueda A, Rizzi N, Benitah JP, Colombi B, Napolitano C, et al Increased Ca2+ sensitivity of the ryanodine receptor mutant RyR2-R4496C underlies cate- cholaminergic polymorphic ventricular tachycardia Circ Res 2009;104:201–9.

38 Cerrone M, Noujaim SF, Tolkacheva EG, Talkachou A, O’Connell R, Berenfeld O, et al Arrhythmogenic mechanisms in a mouse model of catecholaminergic polymorphic ventri- cular tachycardia Circ Res 2007;101:1039–48.

39 Paavola J, Viitasalo M, Laitinen-Forsblom PJ, Pasternack M, Swan H, Tikkanen I, et al Mutant ryanodine receptors in catecholaminergic polymorphic ventricular tachycardia gener- ate delayed afterdepolarizations due to increased propensity to Ca 2+ waves Eur Heart J 2007; 28:1135–42.

40 Bers DM Macromolecular complexes regulating cardiac ryanodine receptor function J Mol Cell Cardiol 2004;37:417–29.

41 Rizzi N, Liu N, Napolitano C, Nori A, Turcato F, Colombi B, et al Unexpected structural and functional consequences of the R33Q homozygous mutation in cardiac calsequestrin: a complex arrhythmogenic cascade in a knock in mouse model Circ Res 2008;103:298–306.

42 Priori SG, Napolitano C, Memmi M, Colombi B, Drago F, Gasparini M, et al Clinical and molecular characterization of patients with catecholaminergic polymorphic ventricular tachy- cardia Circulation 2002;106:69–74.

43 Rossenbacker T, Bloise R, De Giuli L, Raytcheva-Buono EV, Theilade J, Keegan R, et al Catecholaminergic polymorphic ventricular tachycardia: genetics, natural history and response

to therapy Circulation 2007;116(Suppl II):179.

44 Hayashi M, Denjoy I, Extramiana F, Maltret A, Buisson NR, Lupoglazoff JM, et al Incidence and risk factors of arrhythmic events in catecholaminergic polymorphic ventricular tachy- cardia Circulation 2009;119:2426–34.

45 Cerrone M, Colombi B, Bloise R, Memmi M, Moncalvo C, Potenza D, et al Clinical and molecular characterization of a large cohort of patients affected with catecholaminergic polymorphic ventricular tachycardia Circulation 2004;110(Suppl):552.

46 Aizawa Y, Komura S, Okada S, Chinushi M, Aizawa Y, Morita H, et al Distinct U wave changes in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT) Int Heart J 2006;47(3):381–9.

47 Postma AV, Denjoy I, Kamblock J, Alders M, Lupoglazoff JM, Vaksmann G, et al aminergic polymorphic ventricular tachycardia: RyR2 mutations, bradycardia, and follow up

Catechol-of the patients J Med Genet 2005;42:863–70.

48 Priori SG, Napolitano C, Schwartz PJ, Grillo M, Bloise R, Ronchetti E, et al Association of long QT syndrome loci and cardiac events among patients treated with beta-blockers J Am Med Assoc 2004;292:1341–4.

49 Sumitomo N, Harada K, Nagashima M, Yasuda T, Nakamura Y, Aragaki Y, et al aminergic polymorphic ventricular tachycardia: electrocardiographic characteristics and optimal therapeutic strategies to prevent sudden death Heart 2003;89:66–70.

Catechol-50 Liu N, Ruan Y, Priori SG Catecholaminergic polymorphic ventricular tachycardia Prog Ccardiovasc Dis 2008;51:23–30.

51 Monteforte N, Raytcheva-Buono E, Bloise R, Shakibi J, Correia MJ, Gasparini M, et al Electrocardiographic analysis of arrhythmias developing during exercise in patients with catecholaminergic polymorphic ventricular tachycardia Circulation 2007;116(II):492.

52 Ormaetxe JM, Saez R, Arkotxa MF, Martinez-Alday JD Catecholaminergic polymorphic ventricular tachycardia detected by an insertable loop recorder in a pediatric patient with exercise syncopal episodes Pediatr Cardiol 2004;25:693–5.

53 Hasdemir C, Priori SG, Overholt E, Lazzara R Catecholaminergic polymorphic ventricular tachycardia, recurrent syncope, and implantable loop recorder J Cardiovasc Electrophysiol 2004;15:729.

54 Rosen MR, Danilo Jr P Effects of tetrodotoxin, lidocaine, verapamil, and AHR-2666 on Ouabain-induced delayed afterdepolarizations in canine Purkinje fibers Circ Res 1980;46: 117–24.

55 Swan H, Laitinen P, Kontula K, Toivonen L Calcium channel antagonism reduces induced ventricular arrhythmias in catecholaminergic polymorphic ventricular tachycardia patients with RyR2 mutations J Cardiovasc Electrophysiol 2005;16:162–6.

exercise-56 Rosso R, Kalman JM, Rogowski O, Diamant S, Birger A, Biner S, et al Calcium channel blockers and beta-blockers versus beta-blockers alone for preventing exercise-induced arrhythmias in catecholaminergic polymorphic ventricular tachycardia Heart Rhythm 2007; 4:1149–54.

57 Watanabe H, Chopra N, Laver D, Hwang HS, Davies SS, Roach DE, et al Flecainide prevents catecholaminergic polymorphic ventricular tachycardia in mice and humans Nat Med 2009; 15(4):380–3.

58 Wilde AA, Bhuiyan ZA, Crotti L, Facchini M, De Ferrari GM, Paul T, et al Left cardiac sympathetic denervation for catecholaminergic polymorphic ventricular tachycardia N Engl J Med 2008;358:2024–9.

59 Scott PA, Sandilands AJ, Morris GE, Morgan JM Successful treatment of catecholaminergic polymorphic ventricular tachycardia with bilateral thoracoscopic sympathectomy Heart Rhythm 2008;5:1461–3.

60 Wehrens XH, Lehnart SE, Reiken SR, Deng SX, Vest JA, Cervantes D, et al Protection from cardiac arrhythmia through ryanodine receptor-stabilizing protein calstabin2 Science 2004; 304:292–6.

61 Wu Y, Roden DM, Anderson ME Calmodulin kinase inhibition prevents development of the arrhythmogenic transient inward current Circ Res 1999;84:906–12.

62 Liu N, Colombi B, Napolitano C, Priori S Ca2+/calmodulin kinase II inhibitor abolishes and prevents triggered activity in myocytes from RyR2R4496C+/-knock-in mice Heart Rhythm 2007;4(Suppl):S107–8.

63 Catterall WA Structure and regulation of voltage-gated Ca2+ channels Annu Rev Cell Dev Biol 2000;16:521–55.

64 Hanlon MR, Wallace BA Structure and function of voltage-dependent ion channel regulatory

68 Splawski I, Timothy KW, Decher N, Kumar P, Sachse FB, Beggs AH, et al Severe arrhythmia disorder caused by cardiac L-type calcium channel mutations Proc Natl Acad Sci USA 2005;102:8089–96.

69 Reichenbach H, Meister EM, Theile H The heart–hand syndrome A new variant of disorders

of heart conduction and syndactylia including osseous changes in hands and feet Kinderarztl Prax 1992;60:54–6.

70 Marks ML, Whisler SL, Clericuzio C, Keating M A new form of long QT syndrome associated with syndactyly J Am Coll Cardiol 1995;25:59–64.

71 Brugada P, Brugada J Right bundle branch block, persistent ST segment elevation and sudden cardiac death: a distinct clinical and electrocardiographic syndrome A multicenter report.

J Am Coll Cardiol 1992;20:1391–6.

72 Antzelevitch C, Brugada P, Borggrefe M, Brugada J, Brugada R, Corrado D, et al Brugada syndrome: report of the second consensus conference: endorsed by the Heart Rhythm Society and the European Heart Rhythm Association Circulation 2005;111:659–70.

21 Intracellular Calcium Handling and Inherited Arrhythmogenic Diseases 407

73 Priori SG, Napolitano C, Gasparini M, Pappone C, Della Bella P, Giordano U, et al Natural history of Brugada syndrome: insights for risk stratification and management Circulation 2002;105:1342–7.

74 Chen Q, Kirsch GE, Zhang D, Brugada R, Brugada J, Brugada P, et al Genetic basis and molecular mechanism for idiopathic ventricular fibrillation Nature 1998;392:293–6.

Chapter 22

Channel Dysfunction in LQT3 Syndrome

Thomas Zimmer and Klaus Benndorf

22.1 Introduction

Voltage-gated sodium channels (VGNC) mediate the fast upstroke of action tials (APs) in electrically excitable cells by rapidly increasing the Na+permeability[1] These channels are heteromultimeric proteins consisting of a large pore-forming a subunit and small accessory b subunits Ten different a and four bsubunit isoforms have been cloned from different mammalian tissues [2] The asubunit is composed of four homologous domains (DI–DIV) that are connected byintracellular linkers (Fig.22.1a) Each domain contains six transmembrane span-ning segments (S1–S6) The S4 regions are essential structural elements of thevoltage sensor They carry regularly arranged positive charges that respond to adepolarizing voltage pulse by a transient outward movement, thereby initiating theopening of the pore The pore is formed by S5 and S6 segments, and the connectingextracellular loops, the so-called P loops These P loops contain key residues forion selectivity and tetrodotoxin binding [1] Images derived from cryo-electronmicroscopy provided fascinating insight into the 3-D structure of a VGNC [3] Thechannel is bell-shaped and forms a central pore that is connected to the intra- andextracellular sides by four separate branches

poten-The Na+channel isoform Nav1.5, encoded by theSCN5A gene, is the nant a subunit in the heart and plays a key role for excitability of atrial andventricular cardiomyocytes and for rapid impulse propagation through the conduc-tion system [4,5] Electrophysiological and biochemical studies provided strongevidence in support of the expression of neuronal and skeletal muscle Na+channels

predomi-in the heart [6] However, the functional significance of these isoforms for thehuman heart is still a matter of debate Mutations in neuronal and skeletal muscleisoforms have not yet been linked to cardiac diseases In contrast, mutations inSCN5A can cause a broad variety of pathophysiological phenotypes (Table22.2)

T Zimmer ( *) and K Benndorf

Institute of Physiology II, University Hospital Jena, Friedrich Schiller University Jena, gasse 9, Jena 07743, Germany

Kollegien-e-mail: thomas.zimmer@mti.uni-jena.de

O.N Tripathi et al (eds.), Heart Rate and Rhythm,

DOI 10.1007/978-3-642-17575-6_22, # Springer-Verlag Berlin Heidelberg 2011 409

Fig 22.1 Structure and function of the cardiac voltage-gated Na + channel (a) Proposed brane topology of Nav1.5 and important structural elements controlling channel gating Each of the four domains (DI–IV) is composed of six transmembrane helices The fourth segment (S4) contains regularly arranged positive charges that are essential elements of the voltage sensor The intracellular loop between DIII and DIV forms the inactivation gate with the key residues isoleucine, phenylalanine, and methionine (IFM motif) [ 1 ] The proximal C terminus is composed

mem-of six helices (H1–H6) [ 18 , 19 ] H1–H4 form an EF-hand domain involved in the binding of Ca++, CaM and the downstream CaM binding motif (IQ motif or H6) [ 16 , 19 , 40 , 41 ] The linker between DIVS6 and H1 is very flexible, but highly conserved among Na+channels, implicating an essential role for proper interaction of the C terminus with other intracellular channel regions during inactivation [ 19 ] (b) Functional states of voltage-gated Na+channels (c) Schematic time rela- tionship between ECG and ventricular action potential in normal (solid line) and LQT3 hearts (dotted line) The control QT interval corrected for heart rate (QTc) is <460 ms The mean QTc in the LQT3 cases listed in Table 22.2 is 542.5
82 ms (s.d.; n ¼ 26 mutations)

410 T Zimmer and K Benndorf

Mutations can either result in gain-of-function or loss-of-function defects, ing on whether Na+entry into the cell is enhanced or reduced, respectively Bothgain-of-function and loss-of-function can be caused by an alteration of severaldistinct channel properties Consequently, a single point mutation can also lead to

depend-a combindepend-ation of both types of gdepend-ating defects (Tdepend-able22.2), although such mutationsare rare

The most important SCN5A channelopathies are long QT syndrome type 3(LQT3) and Brugada syndrome (BrS) LQT3 is caused exclusively by SCN5Amutations, whereas BrS has been associated withSCN5A mutations in less than20% of the patients [7] The other cardiac excitation abnormalities, listed inTable 22.2, are anything but rare However, point mutations in SCN5A as theprimary cause of these diseases are more the exception than the rule For example,sudden infant death syndrome (SIDS) is a fatal event that can result from variousorigins, like position in crib, location of pillow, passive smoking, nonbreast feed-ing, or mutations in several genes including the cardiac K+channelsKCNQ1 andKCNH2 [8].SCN5A mutations detected in SIDS victims were investigated uponheterologous expression, and both LQT3- and BrS-type mutant channels wereobserved (Table22.2) However, the situation becomes more complicated whentaking into account that Nav1.5 is regulated by more than ten interacting proteins[9] In the last 3 years already nine distinct cardiac disease phenotypes have beenattributed to mutations in genes coding for Nav1.5 regulatory proteins One canexpect the discovery of a growing number of such mutations by future geneticscreenings in cardiac cases Defects in interacting proteins resulted in gain- or loss-of-function of wild-type Nav1.5, i.e., in defects similar to those observed in mutant

Cardiac conduction disease (CCD) [ 23 , 56 , 57 ] Sick sinus syndrome (SSS) [ 58 , 59 ] Atrioventricular block (AV block) [ 60 ] Atrial standstill (AS) [ 61 ] Atrial fibrillation (AF) [ 54 , 62 , 63 ] Sudden infant death syndrome (SIDS) [ 8 ] Dilated cardiomyopathy (DCM) [ 62 , 64 ]

“SCN5A overlap syndromes” [ 23 , 65 , 66 ]

BrS þ CCD þ SSS [ 67 ] Gain- and loss-of-function “SCN5A overlap syndromes” [ 23 , 65 , 66 ]

This chapter outlines the currently knownSCN5A mutations linked to LQT3 Wesummarize the results on mutant channel function, and we discuss these functionaldata in terms of Na+channel structure–function relationships and in terms of theimpact on QTc intervals The mutations considered herein cannot make up acomplete list as considerably more mutations may have been identified by geneticscreenings but not studied electrophysiologically, published, or included in onlinedatabases.

The gating mechanism in VGNCs can be described by a simple three-state model(Fig.22.1b) At the resting membrane potential, channels are in the closed state.Depolarization of the cell initiates the transition to the open, i.e., the conductingstate Initially, three of the four S4 segments (DI–DIII) respond to the change ofmembrane voltage by a fast transient outward movement across the membrane [1]

It is very likely that these charged S4 helices move along a spiral path through fourdistinct gating pores, visible as peripherally located transmembrane pores in imagesfrom cryo-electron microscopy [1,3] The transition to the inactivated state nor-mally occurs within a few milliseconds of opening Notably, this open-state inacti-vation is coupled to activation and is initiated by the S4 in domain IV [10,11].This positively charged segment displays a delayed response to an external voltagestimulus It moves when the channel already conducts Na+ This deceleratedresponse finally generates the signal for a movement of intracellularly locatedchannel structures, occluding the pore and terminating Na+influx A tight channelclosure is essential for an efficient AP repolarization and termination In wild-typechannels, the fraction of the Na+current, that persists throughout the AP, is less than0.3% of the transient current [12]

Three Na+ channel structures are essentially involved in this fast open-stateinactivation The most important structure is the inactivation gate which is formed

by the DIII–DIV linker This linker contains a cluster of the hydrophobic residuesisoleucine, phenylalanine, and methionine (IFM motif) that are flanked by two ahelices (Fig.22.1a) [13] The IFM motif in the inactivation gate serves as a lid andneighboring glycine and proline residues function as molecular hinges (“hinged-lidmodel”) [1] Inactivation initiates when the inactivation gate moves toward itsreceptor formed by amino acid residues in DIVS6 and in intracellular loopsDIIIS4/S5 and DIVS4/S5, and located within and near the intracellular mouth ofthe pore [1] The third structure involved in channel inactivation is the C terminus[14] Evidence has accumulated that the proximal half of the C terminus function-ally interacts with the DIII–DIV linker, which stabilizes the inactivation gate-occluded channel and minimizes channel reopening [13] This interaction involvescalmodulin (CaM) [15–17], which can bind to a C terminal CaM binding motif(IQ motif or helix H6 in Fig.22.1a) [16] Interestingly, CaM is not the only Ca2+sensor regulating Na 1.5 inactivation The channel also contains an EF-hand

22 Molecular Mechanisms of Voltage-Gated NaþChannel Dysfunction 415

domain, i.e., an intrinsic Ca2+sensor (helices H1–H4 in Fig.22.1a) [17–19] ThisEF-hand domain provides structural elements not only for Ca2+but also for CaM/IQmotif binding [16,19] Together these data indicate that channel inactivation is acomplex process, involving excitation-mediated fluctuations in intracellular [Ca2+],CaM action on different Nav1.5 intracellular structures, as well as Ca2+sensing and

IQ motif binding by an EF-hand domain The detailed sequence of cellular eventsand intramolecular rearrangements in Nav1.5 resulting in correct channel inactiva-tion remains an interesting and challenging field of ongoing research

The recovery from the inactivated state, i.e., the transition back to the closed state(Fig.22.1b), depends on both time and membrane potential In the closed state, theinactivation gate has returned to its initial conformation and the channel is occluded

by pore residues Upon membrane depolarization, Na+channels may open again, oralternatively, they may inactivate without opening (“closed-state inactivation”) Themembrane potential at which half of the cardiac Na+ channels are available foractivation is a few millivolt negative to80 mV [2] A rise in intracellular calciumion concentration ([Ca2+]i) destabilizes the inactivated state, i.e., it shifts the steady-state availability toward depolarized potentials [17,20] The opposite effect is seenwhen antiarrhythmic drugs, like lidocaine, are applied [21,22]

Rapid opening, fast transition to the inactivated state, tight channel closureduring the AP plateau and during the early phase of repolarization are essentialprerequisites for the generation of proper cardiac APs and, consequently, also fornormal QT intervals in the ECG The inherited LQT syndrome is characterized byprolonged ventricular repolarization and increased QT intervals (Fig.22.1c), pre-disposing to torsades de pointes (TdP), ventricular tachyarrhythmias, recurrentsyncope, or even sudden cardiac death (SCD) In LQT3, mutant Nav1.5 channelsinterfere with ventricular repolarization by antagonizing the repolarizing K+cur-rent, i.e., they conduct Na+ ions when wild-type channels are normally closed.Different mutations and mechanisms leading to such a depolarizing effect aredescribed below

To our knowledge, 87 SCN5A mutations are known to be related to LQT3(Fig.22.2a) Eighty of them are missense mutations In seven cases, nucleotidedeletions (n ¼ 6) or insertions (n ¼ 1) caused in-frame modifications of the pro-tein Nonsense mutations have not yet been discovered in LQT3 patients, in contrast

to many BrS mutations leading to loss-of-function Figure 22.2a illustrates thatmost of the LQT3 mutations cluster in intracellular regions and that transmembranesegments as well as extracellular S1/S2 and S3/S4 regions are less frequentlyaffected This conclusion is true in terms of the absolute numbers of mutations(Fig.22.2b) However, it ignores that intracellular regions form more than half ofthe channel protein (1,080 amino acids) [4], whereas transmembrane segments andextracellular S1/S2 plus S3/S4 regions constitute smaller protein parts (totally

551 and 68 amino acids, respectively) Figure 22.2b suggests that these three

416 T Zimmer and K Benndorf

Nav1.5 regions are similarly important for channel gating and inactivation(Fig.22.2b) In contrast, LQT3 mutations were not detected in the P loops con-necting S5 and S6 (totally 317 amino acids), in S1 segments (totally 93 amino

Fig 22.2 SCN5A mutations associated with LQT3 (a) and their incidence in extracellular, transmembrane, and intracellular regions (b) Note that persistent currents are frequently detected

in combination with other gain-of-function features (for details see Table 22.1 ) Q692K was associated with the KCNQ1 mutation R562M [ 42 ] Other references for mutations not yet characterized and thus not listed in Table 22.1 are: [ 43 ] for G9V, R225Q, G639R, S1609W; [ 44 ] for R975W; [ 45 ] for R18W, V125L, Q245K, L404Q, N406K, V411M, E462K, P637L, P648L, R971C, T1069M, E1225K, E1231K, S1458Y, G1481E, R1623L, V1667I, T1779M, Q1909R, A1949S, R1958Q; [ 46 ] for D1114N, L1501V, R1623L, S1787N; [ 47 ] for R190Q; [ 48 ] for A413T, A413E, Q573E, G579R, R689H, M1498T, I1660V, Y1767C, R1913H; [ 49 ] for T1645M; [ 50 ] for D1839G; [ 51 ] for delF1486

22 Molecular Mechanisms of Voltage-Gated NaþChannel Dysfunction 417

acids), or in the transmembrane part of domain 2 (228 amino acids) Consequently,gating defects leading to a clinically relevant gain-of-function can either not beachieved by mutations in these regions or generate lethality during embryonicdevelopment In contrast to LQT3 mutations, the P loops are a preferred targetfor BrS and cardiac conduction disease (CCD) mutations [23].

Only 45 of the 87 mutant channels have been characterized so far by gous expression and electrophysiological measurements Most of them (33 muta-tions) developed an increased persistent Na+current compared to wild-type Nav1.5(see Sect.22.4; Fig.22.2aand Table22.1) For the other 12 mutants, only alterna-tive gain-of-function mechanisms were observed (see Sect 22.5; Fig.22.2aandTable22.1)

heterolo-22.4 Persistent Na+Current: A Characteristic Feature

of Most LQT3 Mutant Channels

A persistent Na+ current is caused by a continuous flow of Na+ through theinactivation-deficient channel during the AP plateau and repolarization phase.Such a sustained current component, schematically illustrated in Fig.22.3a, can

be expected to prolong the ventricular AP by directly counteracting repolarizing K+currents This mechanism is considered as the primary cause of the disease, becausemost LQT3 mutations characterized so far resulted in this inactivation defect(Fig.22.2a) The persistent inward current is still small compared to the transientinward current, indicating that even minor inactivation defects result in seriousclinical consequences For instance, in DKPQ channels that generate some of thelargest mutation-associated persistent currents, the sustained current component isstill smaller than 5% of the transient current [12,24,25]

Because a persistent current results from defective open-state inactivation,LQT3 mutations should indicate amino acid positions that are essential for thetransition to and stabilization of the inactivated state Those mutations indeedcluster in regions known to be involved in open-state inactivation (Fig 22.3a)

An increased persistent current was detected in all LQT3 mutations in the tion gate (DIII–IV linker) and in the proximal part of the C terminus, as well as infive out of nine mutations in known inactivation gate receptor regions (DIIIS4/S5,DIVS4/S5, and DIVS6) Due to the essential role of DIVS4 in coupling Na+channelactivation to fast inactivation, it is conceivable that mutations in this S4 helixalso destabilize the inactivated state Moreover, onset of inactivation should bealso delayed when gating charges are removed Both features, i.e., deceleratedcurrent decay and a sustained inward current, are indeed intrinsic to R1623Q,R1626P, and R1644H [12, 25–28] However, R1644C was also associatedwith BrS [29], and increased persistent currents through mutant R1644C channelswere not reported (Table22.1)

inactiva-A persistent current caused by mutations in DI–DII and DII–DIII linkers indicatesthat these regions are also involved in open-state inactivation The function of these

418 T Zimmer and K Benndorf

linkers in channel inactivation is not yet clear The distal portion of the C terminusmay act in a similar way However, deletion of this region (del1922–2016) did notresult in an elevated persistent current [18], suggesting that it is not necessary for tightchannel closure Mutation S216L, located three amino acid positions upstream of thefirst positively charged arginine in DIS4, is the only known LQT3 mutation in anextracellular region that resulted in a robust persistent Na+current [30].

Published QTc values as a function of the persistent current in correspondingmutant channels are plotted in Fig.22.3b Remarkably, there is no obvious correlation

Fig 22.3 Summary on LQT3 mutations showing enlarged persistent currents (a) Schematic current traces from normally inactivating (solid line) and partially noninactivating (dotted line) channels, and distribution of corresponding mutations over the proposed Nav1.5 structure The size

of the persistent current relative to the transient current is typically between 1 and 3% in LQT3 mutants, and 0.3% in wild-type Nav1.5 (b) Plot of QTc intervals in LQT3 patients versus the relative persistent current in corresponding mutant channels There is no proportional relation between both parameters QTc values and electrophysiological properties of the mutant channels are given in Table 22.1 White dots – one additional gain-of-function mechanism, grey dots – two other gain-of-function mechanisms, black dots – three other gain-of-function mechanisms No other gain-of-function properties were reported for V1777M [ 31 ] The horizontal dotted line represents the borderline QTc value of 460 ms; the triangle indicates the average QTc of healthy controls WT wild-type allele

22 Molecular Mechanisms of Voltage-Gated NaþChannel Dysfunction 419

between the size of this noninactivating current fraction and QTc intervals in patients.QTc can be prolonged even in the absence of a persistent current, while on the otherhand, relatively large persistent currents can be linked to borderline QTc values.There are several reasons for this apparent inconsistency Firstly, several cellular,genetic, and environmental factors may considerably influence the activity of mutantchannels in the intact heart [23] Interacting proteins,SCN5A polymorphisms, xeno-biotics, or body core temperature are examples for such modulatory factors that areoften not considered when expressing mutant channels in a heterologous host.Consequently, severe electrophysiological defects, which could be expected from alife-threatening clinical phenotype, might not be detected Such parameters may evencause variability of the QTc interval among mutant gene carriers of the same family.Second and most importantly, LQT3 is caused by multiple gain-of-function mechan-isms in Nav1.5 (see Sect.22.5below; Table22.1) Mutant channels can be character-ized by one, two, or even three additional gain-of-function mechanisms (white, grey,and black symbols in Fig.22.3b, respectively) Figure 22.3b illustrates that mostsymbols for those mutant channels showing no or small persistent currents, or forthose causing the most extreme QTc prolongations, are either black or grey Further-more, it is obvious that all data points above the dotted line, indicating the borderlineQTc value of 460 ms, represent mutants with at least one alternative gain-of-functionfeature, suggesting that a persistent current alone is not sufficient to generate aclinically relevant LQT3 phenotype It is intriguing to speculate that the cardiac APcan tolerate even a robust persistent current, as long as no other gain-of-functionfeatures additively counteract repolarization This interpretation finds further supportfrom the data of Lupoglazoff and co-workers on V1777M [31] Only the homozygousmutation caused a serious QT prolongation (526 ms), whereas the heterozygousmutation in the parents and siblings of the index patient resulted in normal orborderline QTc intervals (415–442 ms) Heterologously expressed V1777M chan-nels, however, generated a large persistent current (3%) that was reduced by 50%when co-expressing simultaneously wild-type and mutant channels Alternative gain-of-function mechanisms were not reported for this mutation Taken together thesefindings support the intriguing idea that an increased persistent current is one of theQTc-prolonging factors, but does not induce the LQT3 syndrome in the absence ofadditional gating defects.

22.5 Alternative Gain-of-Function Defects in Nav1.5

Mutant Channels

Four alternative mechanisms have been suggested in LQT3 syndrome: (a) increasedwindow current, (b) delayed onset of open-state inactivation resulting in deceler-ated current decay, (c) faster recovery from inactivation, and (d) a higher peakcurrent density As mentioned above, combinations of two or more gain-of-functionfeatures are the rule rather than the exception (Table 22.1) We would like to

420 T Zimmer and K Benndorf

emphasize that several of those alternative gain-of-function defects were alsoobserved in Nav1.5 mutant channels related to atrial fibrillation (AF) (K1493R,Y1795C, and M1875T in [32–34], respectively) However, patients showed normalQTc intervals (K1493R, M1875T).

A window current results from the overlap of the steady-state inactivation andsteady-state activation curves (Fig.22.4a) A small percentage of channels are notinactivated within this voltage range; they are available for activation and thedepolarized membrane potential is sufficient to open them This window currentflows during the repolarization phase of the AP when the membrane slowly re-enters this voltage range In wild-type channels, this voltage range is very narrowand the effect should be minimal (Fig.22.4a) However, LQT3 mutations oftenresult in relative shifts of the steady-state inactivation and activation curves or inincreased slope factors of these curves (i.e., less steep curves) Such alterations ingating can increase both the critical voltage range and the magnitude of theresulting window current (Table22.1) In many LQT3 mutants, the steady-stateinactivation curve is shifted toward depolarized potentials (Fig.22.4a), but steady-state activation is unchanged or shifted to a lesser extent into the same direction.Some of those LQT3 mutations cause critically prolonged QT intervals even inthe absence of a persistent current (e.g., E1295K, A1330P, A1330T, and I1768V)[35–38] It is interesting to note that most of those mutations cluster in structuralelements known to be involved in fast channel closure, except for those located

in the proximal half of the C terminus (Fig.22.4a) Those mutations collectivelyreduced steady-state availability, thus creating a substrate for a loss-of-functiondisease finally resulting in a “SCN5A overlap syndrome” (E1784K, 1795insD;Tables22.1and22.2)

A delayed onset of fast inactivation resulting in a decelerated decay of scopic currents is often observed not only in combination with a persistent current,but occurs also in the absence of a persistent current (Table 22.1, Fig 22.4b)

macro-A slower current decay alone does not necessarily directly prolong the macro-AP, but mayaffect voltage-dependent activity of other outward or inward currents that arecrucial for AP duration Interestingly, several of the LQT3 mutant channels show

a partial loss of the voltage dependency of fast inactivation (current decay), ing not only in accelerated inactivation kinetics at less depolarized potentials, butalso in slowing of current decay at more depolarized potentials (Table 22.1).Channels showing faster current decay kinetics (Fig 22.4b) are characterized byother gain-of-function features

result-A faster recovery from inactivation, the third alternative arrhythmia mechanism,

is found in most LQT3 mutant channels (Fig.22.4c) As shown for I1768V [39],faster recovery from inactivation results in channel re-opening and thus in anincreased Na+ current component during cardiac repolarization, which in turnprolongs the AP A theoretical approach confirmed that a twofold increase in therecovery rate results in nearly a doubling of this inward current [39]

Larger current amplitudes could also prolong the cardiac AP, via an indirecteffect on other ionic currents, or alternatively, via proportionally increased persis-tent currents Currently, it is not clear whether higher current densities are specific

22 Molecular Mechanisms of Voltage-Gated NaþChannel Dysfunction 421

Fig 22.4 Alternative gain-of-function mechanisms in LQT3 mutant channels (a) An increased window or overlap current is caused by relative shifts of the steady-state inactivation and activa- tion curves (dark grey area), as seen in 16 out of the 45 characterized LQT3 mutant channels In most cases, only a depolarizing shift of steady-state inactivation was observed (see Table 22.1 ) Alterations of the steepness of both curves may also affect the window current, but were not considered in this report (b) Slower current decay resulting from decelerated open-state inactiva- tion was reported in 18 different LQT3 mutant channels Interestingly, preferred targets were the S4/S5 linkers in DIII and DIV, which are part of the inactivation gate receptor A reduced voltage dependency of inactivation, i.e., a faster inactivation at less depolarized potentials and a slower inactivation at more depolarized potentials, was preferentially seen when the inactivation gate and the extracellular S3/S4 linker in DIV were mutated (c) Accelerated recovery from inactivation was frequently observed in LQT3 mutant channels (23 out of 45) In 4, 7, and 11 cases, recovery was decelerated, not investigated, or unchanged compared to wild-type Na 1.5 (Table 22.1 ), respectively

422 T Zimmer and K Benndorf

for the heterologous host and whether they are indeed relevant for the patients’phenotype The mutant channels that produced larger current densities also dis-played at least one of the other gain-of-function features (Table22.1).

22.6 Conclusions

We conclude that it is difficult to assess the contribution of an individual function defect for a QTc prolongation, and for the severity of the clinical pheno-type Definitely, LQT3 is caused by multiple gain-of-function gating defects in

gain-of-Nav1.5 mutant channels, and an increased persistent current is only one of the keyQTc prolonging factors However, this inactivation defect seems not to be suffi-cient, suggesting that ventricular cells can somehow tolerate such a late current, atleast in heterozygous mutant gene carriers Moreover, as shown in Table22.1andFig.22.4, some LQT3 mutant channels showed also loss-of-function properties,like faster open-state inactivation or reduced steady-state availability These prop-erties may counteract QT prolongation They may even lead to a diagnosedcombination of a gain-of-function and loss-of-function disease, resulting in a

“SCN5A overlap syndrome”

3 Sato C, Ueno Y, Asai K, Takahashi K, Sato M, Engel A, et al The voltage-sensitive sodium channel is a bell-shaped molecule with several cavities Nature 2001;409:1047–51.

4 Gellens ME, George Jr AL, Chen LQ, Chahine M, Horn R, Barchi RL, et al Primary structure and functional expression of the human cardiac tetrodotoxin-insensitive voltage- dependent sodium channel Proc Natl Acad Sci USA 1992;89:554–8.

5 Blechschmidt S, Haufe V, Benndorf K, Zimmer T Voltage-gated Na+channel transcript patterns in the mammalian heart are species-dependent Prog Biophys Mol Biol 2008;98: 309–18.

6 Haufe V, Chamberland C, Dumaine R The promiscuous nature of the cardiac sodium current J Mol Cell Cardiol 2007;42:469–77.

7 Schulze-Bahr E, Eckardt L, Breithardt G, Seidl K, Wichter T, Wolpert C, et al Sodium channel gene (SCN5A) mutations in 44 index patients with Brugada syndrome: different incidences in familial and sporadic disease Hum Mutat 2003;21:651–2.

8 Otagiri T, Kijima K, Osawa M, Ishii K, Makita N, Matoba R, et al Cardiac ion channel gene mutations in sudden infant death syndrome Pediatr Res 2008;64:482–7.

9 Abriel H Cardiac sodium channel Nav1.5 and interacting proteins: Physiology and physiology J Mol Cell Cardiol 2010;48:2–11.

patho-22 Molecular Mechanisms of Voltage-Gated NaþChannel Dysfunction 423

10 Sheets MF, Kyle JW, Kallen RG, Hanck DA The Na channel voltage sensor associated with inactivation is localized to the external charged residues of domain IV, S4 Biophys J 1999;77:747–57.

11 Chanda B, Bezanilla F Tracking voltage-dependent conformational changes in skeletal muscle sodium channel during activation J Gen Physiol 2002;120:629–45.

12 Dumaine R, Wang Q, Keating MT, Hartmann HA, Schwartz PJ, Brown AM, et al Multiple mechanisms of Na+channel-linked long-QT syndrome Circ Res 1996;78:916–24.

13 Motoike HK, Liu H, Glaaser IW, Yang AS, Tateyama M, Kass RS The Na+ channel inactivation gate is a molecular complex: a novel role of the COOH-terminal domain.

J Gen Physiol 2004;123:155–65.

14 Mantegazza M, Yu FH, Catterall WA, Scheuer T Role of the C-terminal domain in tion of brain and cardiac sodium channels Proc Natl Acad Sci USA 2001;98:15348–53.

inactiva-15 Kim J, Ghosh S, Liu H, Tateyama M, Kass RS, Pitt GS Calmodulin mediates Ca 2+

sensitivity of sodium channels J Biol Chem 2004;279:45004–12.

16 Shah VN, Wingo TL, Weiss KL, Williams CK, Balser JR, Chazin WJ Calcium-dependent regulation of the voltage-gated sodium channel hH1: intrinsic and extrinsic sensors use a common molecular switch Proc Natl Acad Sci USA 2006;103:3592–7.

17 Potet F, Chagot B, Anghelescu M, Viswanathan PC, Stepanovic SZ, Kupershmidt S, et al Functional interactions between distinct sodium channel cytoplasmic domains through the action of calmodulin J Biol Chem 2009;284:8846–54.

18 Cormier JW, Rivolta I, Tateyama M, Yang AS, Kass RS Secondary structure of the human cardiac Na+channel C terminus: evidence for a role of helical structures in modulation of channel inactivation J Biol Chem 2002;277:9233–41.

19 Chagot B, Potet F, Balser JR, Chazin WJ Structure of the C-terminal EF-hand domain of human cardiac sodium channel Nav1.5 J Biol Chem 2009;284:6436–45.

20 Biswas S, DiSilvestre D, Tian Y, Halperin VL, Tomaselli GF Calcium-mediated dual-mode regulation of cardiac sodium channel gating Circ Res 2009;104:870–8.

21 Nuss HB, Kambouris NG, Marba´n E, Tomaselli GF, Balser JR Isoform-specific lidocaine block of sodium channels explained by differences in gating Biophys J 2000;78:200–10.

22 Hanck DA, Nikitina E, McNulty MM, Fozzard HA, Lipkind GM, Sheets MF Using lidocaine and benzocaine to link sodium channel molecular conformations to state- dependent antiarrhythmic drug affinity Circ Res 2009;105:492–9.

23 Zimmer T, Surber R SCN5A channelopathies – an update on mutations and mechanisms Prog Biophys Mol Biol 2008;98:120–36.

24 Bennett PB, Yazawa K, Makita N, George Jr AL Molecular mechanism for an inherited cardiac arrhythmia Nature 1995;376:683–5.

25 Wang DW, Yazawa K, George Jr AL, Bennett PB Characterization of human cardiac Na+channel mutations in the congenital long QT syndrome Proc Natl Acad Sci USA 1996;93: 13200–5.

26 Kambouris NG, Nuss HB, Johns DC, Tomaselli GF, Marban E, Balser JR Phenotypic characterization of a novel long-QT syndrome mutation (R1623Q) in the cardiac sodium channel Circulation 1998;97:640–4.

27 Makita N, Shirai N, Nagashima M, Matsuoka R, Yamada Y, Tohse N, et al A de novo missense mutation of human cardiac Na+channel exhibiting novel molecular mechanisms of long QT syndrome FEBS Lett 1998;423:5–9.

28 Ruan Y, Liu N, Bloise R, Napolitano C, Priori SG Gating properties of SCN5A mutations and the response to mexiletine in long-QT syndrome type 3 patients Circulation 2007;116: 1137–44.

29 Frustaci A, Priori SG, Pieroni M, Chimenti C, Napolitano C, Rivolta I, et al Cardiac histological substrate in patients with clinical phenotype of Brugada syndrome Circulation 2005;112:3680–7.

30 Wang DW, Desai RR, Crotti L, Arnestad M, Insolia R, Pedrazzini M, et al Cardiac sodium channel dysfunction in sudden infant death syndrome Circulation 2007;115:368–76.

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